Genomic Correlates of Immune-Cell Infiltrates in Colorectal Carcinoma Marios Giannakis, Xinmeng Jasmine Mu, Sachet A. Shukla, Zhi Rong Qian, Ofir Cohen, Reiko Nishihara, Samira Bahl, Yin Cao, Ali Amin-Mansour, Mai Yamauchi, Yasutaka Sukawa, Chip Stewart, Mara Rosenberg, Kosuke Mima, Kentaro Inamura, Katsuhiko Nosho, Jonathan A. Nowak, Michael S. Lawrence, Edward L. Giovannucci, Andrew T. Chan, Kimmie Ng, Jeffrey A. Meyerhardt, Eliezer M. Van Allen, Gad Getz, Stacey B. Gabriel, Eric S. Lander, Catherine J. Wu, Charles S. Fuchs, Shuji Ogino, Levi A. Garraway  Cell Reports  Volume 15, Issue 4, Pages 857-865 (April 2016) DOI: 10.1016/j.celrep.2016.03.075 Copyright © 2016 The Authors Terms and Conditions

Cell Reports 2016 15, 857-865DOI: (10.1016/j.celrep.2016.03.075) Copyright © 2016 The Authors Terms and Conditions

Figure 1 Overview of the Integrative Molecular Epidemiology Approach Employed in the NHS and HPFS Cohorts Participant follow-up used biennial questionnaires. Patients who developed CRC underwent resection of tumor and adjacent normal tissue, followed by formalin fixation, pathologic characterization, and WES. Cell Reports 2016 15, 857-865DOI: (10.1016/j.celrep.2016.03.075) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Long Tail of Significantly Mutated Genes in Non-hypermutated CRC Dark blue: genes identified in the NHS and HPFS but also previously reported in other large-scale sequencing studies of CRC (Cancer Genome Atlas Network, 2012; Seshagiri et al., 2012; Lawrence et al., 2014). Red: genes identified in the NHS and HPFS only. Common protein function categories and major CRC signaling pathways are color coded next to significantly mutated genes. Gray bars, lower panel: log10 q value. Dashed line indicates q = 0.1. Cell Reports 2016 15, 857-865DOI: (10.1016/j.celrep.2016.03.075) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Breakdown of the Total Number of Somatic Mutations and Predicted Neoantigens by Mutation Type Cell Reports 2016 15, 857-865DOI: (10.1016/j.celrep.2016.03.075) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Correlation of Neoantigen Load with Immune-Cell Infiltration in CRC (A) Correlation of neoantigen load with overall lymphocytic reaction score. The dotted line denotes a smoothing curve for all data points. The p value is calculated by Spearman’s rank correlation test. (B) Correlation of neoantigen load with individual components of lymphocytic reaction. The median number of neoantigens for each component score is plotted. The p value is calculated by Spearman’s rank correlation test. (C) Correlation between neoantigen load and density of immunohistochemically defined tumor-infiltrating T cell subsets. Numbers inside bars correspond to the Spearman’s rank correlation coefficient. The p value is calculated by Spearman’s rank correlation test. (D) Comparison of number of neoantigens among tumors with different TIL grades, with analysis restricted to the MSS POLE-wild-type subset. The p value is calculated by Wilcoxon rank-sum test. Cell Reports 2016 15, 857-865DOI: (10.1016/j.celrep.2016.03.075) Copyright © 2016 The Authors Terms and Conditions

Figure 5 Neoantigen Load and Survival in CRC (A and B) Kaplan-Meier curve for (A) CRC-specific survival and (B) overall survival according to neoantigen load. High (n = 149) and medium-low (n = 448) neoantigen groups were classified as described in Experimental Procedures. The p value is calculated by log rank test. Cell Reports 2016 15, 857-865DOI: (10.1016/j.celrep.2016.03.075) Copyright © 2016 The Authors Terms and Conditions

Figure 6 Positive Selection for HLA and APM Mutations in Immune-Cell-Infiltrated Tumors (A) Enrichment of HLA mutations in CRCs with higher TIL scores. The p value is calculated by the chi-square test. (B) Enrichment of HLA mutations in MSS POLE-wild-type CRCs with higher TIL scores. The p value is calculated by the chi-square test. (C) Enrichment of APM mutations in immune-cell-infiltrated tumors. Highlighted genes are representative components of the APM pathway, which are mutated in NHS and HPFS tumors. Numbers in parentheses indicate mutation frequencies (percentage) in non-infiltrated versus infiltrated tumors. Neopeptides are processed by the proteasome, transported into the ER, and loaded onto MHCs that, through vesicle transport, traffic to the plasma membrane of tumor cells, where they can be recognized by effector T cells. Cell Reports 2016 15, 857-865DOI: (10.1016/j.celrep.2016.03.075) Copyright © 2016 The Authors Terms and Conditions

Figure 7 Model of Positive Selection for HLA and APM Mutations in Tumors under Immune Attack Cell Reports 2016 15, 857-865DOI: (10.1016/j.celrep.2016.03.075) Copyright © 2016 The Authors Terms and Conditions